PROJECT SUMMARY/ABSTRACT Coxiella burnetii's pathogenicity depends on its ability to grow in a lysosome-derived hostile vacuole within human cells. However, metabolic processes critical to C. burnetii's intracellular growth are largely unknown. This lack of knowledge has prevented both the understanding of its basic biology and pathogenesis, and the development of better therapeutic agents. The long-term goal is to understand the molecular details of Coxiella's distinctive physiology, and to apply this knowledge to developing novel therapeutic strategies. The objective of this application is to identify metabolic pathways that are vital to C. burnetii's intracellular growth. The central hypothesis, which was formulated based on preliminary data, is that C. burnetii evolved from a tick- associated ancestor by acquiring critical metabolic genes through horizontal gene transfer (HGT). A novel evolutionary genomics approach will be used to identify metabolic pathways that are critical to C. burnetii's intra- cellular growth. The rationale for the proposed research is that once metabolic processes important to C. burnetii's intracellular growth are identified, pharmacological agents that block these pathways could be developed to treat chronic infections more effectively. The objective of this project will be accomplished by three specific aims: (1) Identify metabolic pathways that distinguish C. burnetii from tick-associated Coxiella. The working hypothesis is that genes critical to C. burnetii's intracellular physiology will not be present in avirulent tick- associated Coxiella. The genome of a closely related Coxiella from the tick Ornithodoros rostratus will be sequenced and compared to C. burnetii's genome. (2) Define metabolic pathways that are critical to C. burnetii's intracellular growth. The working hypothesis is that genes acquired via HGT are being maintained in C. burnetii because they are critical to the pathogen's physiology. Phylogenetic approaches will be used to identify HGT-derived genes, and their functions will be validated using RNA-seq and genetic tools. (3) As a proof of principle, determine the importance of heme biosynthesis to C. burnetii's intracellular growth. The working hypothesis is that heme biosynthesis is crucial to Coxiella's growth. Heme production and intracellular growth of heme pathway-deficient strains will be assayed. This study is innovative because it (a) uses a novel approach that overcomes the current limitations in studying Coxiella at a genome-wide scale, and (b) is based on a novel concept that the human pathogen evolved from a tick symbiont via massive HGT. The proposed project is significant because it will (a) uncover metabolic pathways that are critical to the pathogen's intracellular growth, (b) identify new therapeutic targets, for example, HemA and HemL are essential for heme biosynthesis in C. burnetii but are not present in humans cells, (c) provide a model approach for identifying genes involved in host adaptation, which could be applied broadly to other pathogens such as Francisella and Rickettsia spp. where avirulent tick symbionts could be compared to virulent human-specialized strains.